A major energy conversion process in living organisms involves coupling the burning of reduced fuels by respiratory chains to the synthesis of ATP by F1F0 ATP synthases. The enzymes that catalyze these reactions are found embedded in the membranes of mitochondria, chloroplasts, and bacteria, and a transmembrane electrochemical proton gradient appears to function as a required intermediate. The F0 sector of the synthase is composed of membrane spanning subunits (a B2 c12 in E. coli) which conduct protons across the membrane, whereas the F1 sector (alpha3 beta3 gamma delta epsilon) is an extrinsic complex that contains the catalytic sites for ATP synthesis. It has been proposed that proton transport is coupled to ATP synthesis by the rotation of a complex of subunits (gamma epsilon c12) that extends through F0F1. Characterizing this subunit rotation and elucidating other aspects of the mechanism of the synthase are the goals of this proposal. The existence of subunit rotation in F1 is well established but the details of rotational coupling are not well defined. In the proposed studies, a spectral assay will be developed to monitor subunit rotation in fully-active, soluble F1. The approach involves positioning a single fluorescent reporter group on the gamma subunit and a quencher on two of the three catalytic subunits (beta) such that rotation of gamma relative to beta will bring the fluorescent group close to the quenchers to cause transient quenching. This assay will be used to test the kinetic competency of subunit rotation as a required step in ATP synthesis that is coupled to subunit rotation. In contract to F1, subunit rotation in F0 has not been demonstrated. For this purpose, an intersubunit disulfide crosslinking approach will be developed which involves substituting a cys on a c subunit in the c12 oligomer. The test for subunit rotation will be to determine whether the c subunit which is initially in the proper orientation to form a crosslink to subunit a is the same one that occupies that position after catalytic turnover, or whether subunit rotation has moved a different c subunit into that position.